Heterotrimeric G proteins couple cell surface receptors to intracellular effectors at the cytoplasmic face of membranes. Receptor activation of the G proteins a subunits causes the a subunit to release GDP, bind GTP and attain an active conformation. Deactivation is accomplished by GTP hydrolysis. RGS proteins (Regulators of G protein Signaling) interact with G protein a subunits to increase their GTPase activity. This recently discovered family of proteins (>15 proteins) have 3 highly conserved regions. Their amino terminii have the most divergence in both length and composition. A member of the family, the GAIP protein, possesses a cysteine-rich region at the amino terminus, analogous to those in cysteine string proteins. Cysteine string proteins are a family of proteins found on synaptic vesicles in several species and undergo palmitoylation on multiple cysteines. Palmitoylation is the reversible addition of 16 carbon palmitate to cysteine residues through a thioester bond. Palmitoylation occurs on most a subunits and aids their membrane localization. We found that GAIP undergoes palmitoylation probably on multiple cysteines. Palmitoylation was only found on the membrane-associated GAIP suggesting that membrane anchoring occurs through palmitoylation. Since G proteins a subunits are located at membranes, we investigated whether other RGS proteins underwent palmitoylation to increase their hydrophobicity and membrane attachment. RGSr, a retinal RGS protein, is primarily localized to the membrane and undergoes palmitoylation probably on two cysteine residues at the amino terminus. RGS4, a protein homologous to RGSr, is primarily soluble and if palmitoylation occured in the membrane associated protein, it was not detectable. RGS2 does not undergo palmitoylation but is found in the membrane fractions suggesting that multiple mechanisms are involved in anchoring the proteins to membranes. To assess the functional significance of palmitoylation, we needed to obtain purified, palmitoylated protein. Using a cell-free system, both RGSr and RGS4 expressed and purified from E. coli underwent in vitro palmitoylation. Further studies with these proteins are pending.